Thymidylate synthase protein expression by IHC and gene copy number by SISH correlate and show great variability in non-small cell lung cancer

Abstract

Introduction: Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to TS-targeting drugs, e.g., pemetrexed.

Methods: TS protein expression (PE) and gene copy number (GCN) were assayed using immunohistochemistry and silver in situ hybridization, respectively, on primary tumors of 189 resected non-small cell lung patients. Associations with pathological and clinical features and prognosis were explored.

Results: Median immunohistochemistry H-score was 220 (range, 10-380) on a 0 to 400 scale; 17% of the patients had a TS expression of 300 or more. TS PE expression did not significantly differ by histology and did not significantly associate with disease-free survival (DFS) or overall survival (OS). However, there was a tendency for increased DFS (p = 0.12) and OS (p = 0.12) in PE positive (>median) squamous-cell carcinoma (SCC) patients. Median GCN was 2.5 genes/nucleus (range, 1.4-9.6); 29% of patients had GCN of 3 or more, 7% of 4 or more and 0.8% amplification. GCN differed by histology (p = 0.015); 50% of SCCs having GCN more than 2.5 versus 32% of adenocarcinomas. There was no significant relationship between TS GCN and DFS or OS; however, a trend toward better DFS (p = 0.18) and OS (p = 0.10) with increased GCN in SCCs was observed. TS GCN was significantly correlated with PE (r = 0.30, p = 0.0009).

Conclusions: TS PE and GCN vary widely in non-small cell lung and correlate significantly with each other. TS GCN is higher in SCCs, whereas TS PE does not associate with histological subtypes, clinical features, or survival. Variability of TS PE and GCN may indicate potential benefit from pemetrexed therapy in selected SCC patients.

Figure 1

Representative photomicrographs of the varying range of TS protein expression detected by IHC. A) H-score of 70, B) H-score of 150, C) H-score of 210, D) H-score of 280, E) H-score of 355.

Figure 2

Representative photomicrographs of TS gene copy number detected by SISH. A) 1.8 genes per nucleus, B) 3.2 genes per nucleus, C) 3.7 genes per nucleus, D) 5.0 genes per nucleus, E) 9.6 genes per nucleus.

Figure 3

Histograms showing the distribution, frequency, median, range, mean, and standard deviation for TS protein expression in NSCLC. A) entire evaluable population, B) adenocarcinoma, C) squamous cell carcinoma, D) large cell carcinoma or not otherwise specified histology.

Figure 4

Kaplan-Meier survival probability curves. Disease-free survival (A) and overall survival (B) according to TS protein expression for the entire evaluable population stratified at the median (H-score 220). Disease-free survival (C and E) and overall survival (D and F) for those with squamous cell carcinoma and non-squamous carcinoma, respectively, stratified at H-score 220.

Figure 5

Histograms showing the distribution, frequency, median, range, mean, standard deviation and amplification for TS gene copy number in NSCLC. A) the entire evaluable population, B) those with adenocarcinoma, C) squamous cell carcinoma, or D) large cell carcinoma or not otherwise specified histology.

Figure 6

Kaplan-Meier survival probability curves. Disease-free survival (A) and overall survival (B) according to TS gene copy number for the entire evaluable population stratified at the median (GCN 2.5). Disease-free survival (C and E) and overall survival (D and F) for those with squamous cell carcinoma and non-squamous carcinoma, respectively, stratified at GCN 2.5.

Figure 7

Dot plot showing the correlation between TS protein expression, represented by the H-score, and TS gene copy number.